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1 Department of Bacterial Pathogenesis and Infection Control, National Institute of Infectious Diseases, Musashimurayama, Tokyo 208-0011, Japan
2 Laboratory of Microbiology and Immunology, Graduate School of Health Sciences, Tokyo Medical and Dental University, Tokyo 113-8519, Japan
Correspondence
Tsuyoshi Kenri
kenri{at}nih.go.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Pharmaceuticals and Medical Devices Agency, 3-3-2 Kasumigaseki, Chiyoda-ku, Tokyo 100-0013, Japan.
Two supplementary tables, listing the oligonucleotides used in this study, are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Waites et al., 2005). As parasitic bacteria, mycoplasmas can continue to colonize their host even in the presence of specific immune responses. The molecular mechanisms responsible for this immune evasion are not fully understood; however, a number of recent studies have suggested probable mechanisms for continuous infection of mycoplasmas; these include molecular mimicry of host cell components (Jacobs et al., 1995), modulation of host immunity by mycoplasmal cell components (Rottem, 2003), invasion of host cells (Baseman et al., 1996) and generation of surface antigen variants of mycoplasmas (Denison et al., 2005; Rosengarten et al., 2000). Among these strategies, surface antigenic variation is a commonly observed phenomenon in many mycoplasma species (Citti et al., 2005; Yogev et al., 2002). Surface variations may play important roles in interaction between mycoplasmas and host cells during infection. In most of the known cases, the variable surface molecules of mycoplasmas are lipoproteins. Depending upon the species, these lipoproteins are encoded by single or multiple genes and undergo frequent phase and size variation during mycoplasma growth (Citti et al., 2005). Various genetic mechanisms are used to modulate the expression of these lipoprotein genes. These mechanisms include DNA rearrangements (Glew et al., 2002; Shen et al., 2000), nucleotide insertions and deletions in the gene regions (Glew et al., 1998; Yogev et al., 1991), gene conversions (Noormohammadi et al., 2000), and site-specific DNA recombinations (Chopra-Dewasthaly et al., 2008; Lysnyansky et al., 2001). Characterization of these genetic mechanisms may provide us with not only a detailed understanding of the surface antigenic variation mechanism of mycoplasmas but also an insight into the survival strategy of minimalist bacteria through utilization of their small genomes.
Mycoplasma penetrans was first isolated from a urine sample of a human immunodeficiency virus (HIV)-infected patient (Lo et al., 1992). It was also isolated as a potential aetiological agent from a primary antiphospholipid syndrome patient without HIV infection (Yáñez et al., 1999). Although the pathogenicity of M. penetrans to humans remains questionable, the characteristics of this bacterium (i.e. invasion of eukaryotic cells, toxicity to chick embryo, and haemolytic and haemoxidative activity) (Girón et al., 1996; Kannan & Baseman, 2000; Lo et al., 1993) suggest its potential pathogenicity to humans. M. penetrans also has the ability to change its surface antigenicity frequently by changing the expression pattern of the P35 family lipoproteins (Neyrolles et al., 1999; Röske et al., 2001). The P35 family lipoproteins are encoded by the mpl genes and, thus far, 38 mpl genes have been found in the M. penetrans HF-2 genome. Of these 38 genes, 30 form a large gene cluster in a 50 kb region of the genome (Horino et al., 2003; Sasaki et al., 2002). M. penetrans uses a unique mechanism to modulate the expression of these mpl genes. Most of the mpl genes have an independent promoter for their expression. These promoter sequences are present in approximately 135 bp DNA regions and are flanked by 12 bp inverted repeats (IRs). Inversion of these promoters causes ON/OFF switching of individual mpl genes. The invertible promoter elements also contain unique sequences that form a terminator-like structure depending on the direction of promoters (see Fig. 4a). This terminator-like structure may serve to prevent readthrough transcription from preceding genes or antisense transcription from OFF configuration promoters (Horino et al., 2003). Thus, the mpl genes have unique genetic switches, which possess promoter and terminator functions in the short DNA region. To further characterize these unique genetic switches and understand the nature of antigenic variation of M. penetrans, in this study we attempted to identify the factors involved in mpl gene promoter inversions. For this purpose, we reconstructed the mpl promoter inversion system in Mycoplasma pneumoniae and Escherichia coli cells.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and M. pneumoniae strain M129 (Lipman et al., 1969) were cultured in PPLO medium at 37 °C as described previously (Horino et al., 2003; Kenri et al., 2004). Transformation of M. pneumoniae M129 with the staphylococcal transposon Tn4001mod vectors was performed by the electroporation method (Hedreyda et al., 1993). M. pneumoniae transformants were selected in PPLO liquid medium containing 18 µg gentamicin (Gm) ml–1 or 15 µg chloramphenicol (Cm) ml–1. Genomic DNAs of the mycoplasma strains were extracted by the QIAamp mini kit (Qiagen) and were used as templates for PCR to construct the plasmids. E. coli strains were grown in Luria–Bertani (LB) medium with or without 50 µg ml–1 of ampicillin (Ap), kanamycin (Km) or spectinomycin (Sp), or 15 µg Cm ml–1 to select the plasmid markers.
Cloning of the p42 and p35 promoter regions.
PCR primers used for cloning are listed in Supplementary Table S1 (available with the online version of this paper). An approximately 3.2 kb region containing the p42 mpl gene (MYPE6630) and its invertible promoter region (see Figs 1a and 5a) was amplified from M. penetrans HF-2 genomic DNA by PCR with primers p42-F and p42-R. To minimize mutations caused by PCR amplification, a high-fidelity DNA polymerase (PyroBest; TaKaRa) was used. The corresponding region of the p35 mpl gene (MYPE6810) was also amplified from M. penetrans HF-2 by PCR with primers p35-F and p35-R. The amplified p42 and p35 regions were inserted into the pENTR/D-TOPO plasmid by using the TOPO cloning system according to the manufacturer's instructions (Invitrogen), resulting in plasmids pAH501 and pAH502 (Table 1). The cloned p42 and p35 promoter sequences on these plasmids were in the ON direction. To introduce the p42 promoter sequence into M. pneumoniae cells, the cloned p42 region on plasmid pAH501 was transferred to the SmaI site of the Tn4001mod vector plasmid pISM2062.2 (Knudtson & Minion, 1993) by using the Gateway cloning technique (Invitrogen). Briefly, pISM2062.2 was converted into the Gateway destination vector by inserting the Gateway vector conversion system (reading frame cassette A) at the SmaI site. The p42 sequence of the pAH501 plasmid was transferred to the pISM2062.2 destination vector by the LR reaction of the Gateway system. The resulting plasmid was designated pAH511 (Table 1) and introduced into M. pneumoniae M129 by electroporation. The p42 and p35 promoter sequences on the pAH501 and pAH502 plasmids, respectively, were also transferred to the SmaI site of the pCL1920 plasmid, which has a pSC101 replicon (Lerner & Inouye, 1990), by using the Gateway cloning system for introduction into E. coli cells. These plasmids were designated pAH521 and pAH522, respectively (Table 1).
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). The cloned MYPE2900 and MYPE8180 on the pAH301 and pAH302 plasmids were sequenced to confirm that no mutations were incorporated during the PCR cloning. To express MYPE2900 and MYPE8180 in M. pneumoniae cells, the tuf promoter (Ptuf) sequence of M. pneumoniae was linked to MYPE2900 and MYPE8180. The Ptuf sequence was amplified from M. pneumoniae M129 genomic DNA by PCR using primers tuf-F-Bam-TOPO and tuf-R-Afl. The amplified tuf promoter fragment was digested with BamHI and AflIII and ligated into the BamHI–NcoI sites of pAH301 and pAH302, resulting in plasmids pAH303 and pAH304 (Table 1). The MYPE2900 and MYPE8180 genes on the pAH303 and pAH304 (Ptuf-MYPE2900 and Ptuf-MYPE8180, respectively) were then transferred to the SmaI site of the Tn4001mod vector plasmid pKV104 (Hahn et al., 1999) by using the Gateway cloning technique. The resulting Tn4001mod plasmids pAH311 and pAH312 (Table 1) were used to transform M. pneumoniae. To express the MYPE2900 and MYPE8180 genes in E. coli, these genes were amplified from M. penetrans HF-2 genomic DNA by PCR with the primer pairs 2900-F-SacTGG and 2900-R-Xho or 8180F-Nde and 8180-R-Eco, respectively. The amplified MYPE2900 fragments were digested with SacI and XhoI and inserted into the SacI–XhoI site of plasmid pColdI (TaKaRa), resulting in plasmid pAH331mut (Table 1). The MYPE2900mut gene on plasmid pAH331mut was sequenced, and the conversion of the codon UGA to UGG was confirmed (nt position 28–30 of the MYPE2900 gene). The amplified MYPE8180 fragments were digested with NdeI and EcoRI and inserted into the NdeI–EcoRI site of plasmid pColdI, resulting in pAH332 (Table 1). Two UGA codons present in MYPE8180 sequences (at nt positions 145–147 and 517–519) were converted to UGG by using the GeneTailor site-directed mutagenesis system according to the manufacturer's instructions (Invitrogen). After mutagenesis, the nucleotide sequence of the MYPE8180mut gene was confirmed by sequencing. The plasmid carrying the MYPE8180mut gene was designated pAH332mut (Table 1). Plasmids pAH331mut and pAH332mut were introduced into E. coli BL21(DE3) to express MYPE2900 and MYPE8180.
Protein analysis.
E. coli BL21(DE3) strains harbouring plasmids pAH331mut, pAH332mut or pColdI were grown at 37 °C until the mid-exponential phase. The cultures were maintained at 15 °C for 30 min, and 0.2 mM final concentration of IPTG was added. After the addition of IPTG, the cultures were maintained at 15 °C with shaking for 24 h. E. coli cells were collected from a 1 ml volume of the cultures by centrifugation at 20 000 g for 2 min and lysed by adding 150 µl of a sample loading buffer for SDS-PAGE. The samples were then subjected to SDS-PAGE at a load of 15 µl per lane. The proteins were visualized by Coomassie brilliant blue staining. For Western blot analysis, the proteins, separated by SDS-PAGE, were transferred onto a nitrocellulose membrane (Bio-Rad). Monoclonal antibody specific for His6-tag (Cell Signaling Technology) was used at a 1 : 1000 dilution to detect His6-tagged MYPE2900 and MYPE8180 proteins. The reacting antibodies were detected with an alkaline phosphatase-conjugated secondary antibody (goat anti-mouse immunoglobulin G; Promega) and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) Colour Development Substrate (Promega) according to the manufacturer's instructions.
PCR detection of mpl gene promoter inversion.
The PCR primer sets designed for the detection of inversions of the mpl gene promoters are listed in Supplementary Table S2. Each primer set consists of three primers [promoter (P), forward (F) and reverse (R) primers]. Two PCRs were performed using these primer sets (with FP primers or RP primers) for each of the corresponding mpl gene promoters to detect the inversion event (see Figs 1 and 5). Genomic DNAs from M. penetrans HF-2 or M. pneumoniae M129-p42 were analysed with this PCR. The E. coli plasmid DNAs carrying the p42 or p35 promoter regions were also analysed with this method. Genomic DNAs of M. pneumoniae M129-p42 transformants were extracted from 25 ml of culture by the QIAamp mini kit. The genomic DNAs were diluted and 1 ng of each (about 1x106 copies of M. pneumoniae M129 genome) was examined by PCR. For purification of M. penetrans HF-2 genomic DNA, a single colony of M. penetrans HF-2 on a PPLO agar plate [derived from the same culture stock used for genome sequencing (Sasaki et al., 2002)] was picked up and cultured in 2 ml PPLO liquid medium at 37 °C. After growth, 100 µl of the culture was added to 25 ml fresh PPLO liquid medium and cultured until the medium colour changed to orange. Genomic DNA was extracted from 1 ml of the culture by the QIAamp mini kit. One nanogram of the purified genomic DNA (about 6x105 copies of M. penetrans HF-2 genome) was subjected to the PCR analysis. E. coli plasmid DNAs were extracted from 4 ml of the E. coli cultures by the QIAprep Spin Miniprep kit (Qiagen). One microlitre of plasmid solution was subjected to PCR examination. The PCR mixture Premix Ex Taq Hot Start Version (TaKaRa) was used for the PCR examination, and the PCR was performed under the following conditions: 30 cycles of 98 °C for 10 s, 55 °C for 30 s, and 72 °C for 2.5 min. The PCR products were analysed by 0.8 % (w/v) agarose gel electrophoresis and visualized by ethidium bromide staining. For sequencing analysis, the PCR products were excised from agarose gels and extracted by using the MinElute gel extraction kit (Qiagen). The extracted DNAs were sequenced by the dye-termination method by using corresponding P, F, or R primers and an ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Sasaki et al., 2002). The amino acid sequences of MYPE2900 and MYPE8180 share relatively high homologies to the Tsr of Mycoplasma pulmonis and Mycoplasma agalactiae, which were previously shown to mediate site-specific DNA inversions (Chopra-Dewasthaly et al., 2008; Sitaraman et al., 2002). We designed experiments to define whether the MYPE2900 or MYPE8180 products can mediate promoter inversion of mpl genes. Since genetic tools are still unavailable for M. penetrans, an mpl promoter inversion system was reconstructed in M. pneumoniae cells. For this purpose, a 3.2 kb DNA fragment containing the p42 mpl gene and its promoter region of M. penetrans HF-2 was introduced into M. pneumoniae strain M129 by using the staphylococcal transposon Tn4001mod vector, which carries a GmR marker (see Methods). Genomic DNA was extracted from the whole transformed population (M129-p42; the p42 promoter sequence is inserted at random in the chromosome) and was analysed by PCR to examine the direction of the p42 promoter. This PCR analysis (Fig. 1a) gave only the 2 kb product from the genomic DNA of M129-p42 (data not shown), indicating that the direction of the p42 promoter in M129-p42 cells was in the ON direction and that p42 promoter inversion does not occur in M. pneumoniae cells, regardless of the position of the p42 promoter in the M. pneumoniae chromosome.
We then introduced recombinase genes into the M129-p42 cells. The MYPE2900 and MYPE8180 clones linked with M. pneumoniae Ptuf promoter were introduced into M129-p42 cells by using the Tn4001mod derivative vector that carries a CmR marker (see Methods). Thus transformants of M129-p42 (GmR-CmR) having the p42 promoter sequence and the recombinase genes were obtained. We then extracted genomic DNA from these transformants (whole transformed population) and examined the direction of the p42 promoter by PCR. This analysis showed that both 2 and 1.2 kb products were amplified from the genomic DNA carrying the MYPE2900 gene (Fig. 1b, lanes 1 and 2). However, only 2 kb product was obtained from the genomic DNAs carrying the MYPE8180 gene (Fig. 1b, lanes 3 and 4) or Tn4001mod vector alone (Fig. 1b, lanes 5 and 6). These results suggested that the MYPE2900 protein is the recombinase that has an activity to invert the p42 promoter sequence.
Reconstruction of the mpl promoter inversion system in E. coli
To further characterize mpl promoter inversion and to develop a simple experimental system, we reconstructed the mpl promoter inversion system in E. coli. Similar to standard mycoplasmal genes, MYPE2900 and MYPE8180 have UGA codons, which are translated into tryptophan in mycoplasma cells. MYPE2900 has a single UGA codon, and MYPE8180 has two UGA codons. Since these UGA codons block full translation of MYPE2900 and MYPE8180 in E. coli, we converted these UGA codons into UGG. The converted MYPE2900 and MYPE8180 genes (MYPE2900mut and MYPE8180mut) were linked with the E. coli cspA promoter and His6-tag sequence at the 5' end on plasmid pAH331mut and pAH332mut, respectively (see Methods and Table 1). Plasmids pAH331mut and pAH332mut were introduced into E. coli BL21(DE3), and the strain was cultured under appropriate conditions to activate the cspA promoter (see Methods). After 24 h culture at 15 °C with or without IPTG, the expression of MYPE2900 and MYPE8180 proteins was detected by immunoblot analysis specific for His6-tag (Fig. 2). The MYPE2900 and MYPE8180 proteins were produced apparently in the presence of IPTG (Fig. 2, lanes 10 and 12), although the reason for the low expression level of MYPE8180 compared to MYPE2900 is not clear. In addition, a weak expression of MYPE2900 was observed in the absence of IPTG (Fig. 2, lane 9).
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), were introduced into E. coli BL21(DE3) harbouring plasmid pAH321mut or pAH322mut. After culturing these E. coli strains under appropriate conditions to activate the cspA promoter, the plasmids were recovered from the cells and analysed by PCR to determine the directions of the p42 and p35 promoter sequence (see Methods). As shown in Fig. 3, the PCR analysis revealed ON and OFF direction products of the p42 and p35 promoter sequences in the presence of the MYPE2900mut gene (Fig. 3, lanes 1, 2, 3, 4, 9, 10, 11 and 12). The absence of 0.2 mM IPTG did not affect this inversion event (Fig. 3, lanes 1, 2, 9 and 10), suggesting that a small amount of MYPE2900 recombinase was sufficient to catalyse p42 and p35 promoter inversions. On the other hand, only ON direction products were obtained in the presence of the MYPE8180mut gene (Fig. 3, lanes 5, 7, 13 and 15), indicating that the promoters p42 and p35 were locked in the ON direction in these conditions. These results strongly support the conclusion that the MYPE2900 protein is the recombinase that mediates DNA inversions of the mpl gene promoters. We also performed the same experiments in E. coli strain YK1340 (Wada et al., 1988), which lacks the histone-like protein HU. Even in the YK1340 background, inversion of the p42 and p35 promoters occurred only in the presence of the MYPE2900mut gene (data not shown). The recombination sites of DNA inversion (IR sequences) of the p42 and p35 promoters are TAACATAAATTA and TAACAAGAATTA, respectively. The MYPE2900 recombinase can probably recognize both these sequences as a recombination site.
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revealed that nucleotides TAA and ATTA at both ends of 12 bp IRs were conserved; however, five nucleotides in the middle of the repeats differed from each other. The consensus sequence was proposed as TAAYNNNDATTA. To determine whether mpl promoter inversions actually occur at these various IR sequences in M. penetrans cells, we extended the PCR-based inversion detection method. In addition to the primer sets for p42 and p35 promoter regions, we designed eight sets of PCR primers for MYPE6840, 6780, 6740, 6690, 6660, 6560, 6530 and 6490 gene promoters (Supplementary Table S2 and Fig. 5a). Of these 10 promoters, the p42 and p35 promoters were in the ON direction, and the others were in the OFF direction in the consensus genome sequence data of M. penetrans HF-2 (Fig. 5a). To detect the inverted form of these promoters, we analysed M. penetrans HF-2 genomic DNA (1 ng template) by the PCR method with 10 sets of primers (Fig. 5a). This PCR analysis showed that amplification products from both the ON and the OFF direction promoters (approx. 2 and 1 kb products) were obtained from all the 10 promoters examined (Fig. 5b), although the amounts of the ON direction products from MYPE6840 and MYPE6660 promoters and the OFF direction product from MYPE6810 (p35) were low (Fig. 5b, lanes 1, 4 and 11). We analysed the nucleotide sequences of the PCR products of ON and OFF directions and confirmed that inversion of these promoter sequences occurred at the 12 bp IR sequences listed in Fig. 4(b) (data not shown). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Wada et al., 1989). However, these factors may not be essential in mpl promoter inversions. The recombination sites (12 bp IR) of the p42 and p35 genes are similar but slightly different, suggesting that MYPE2900 recombinase has flexibility in recognizing the recombination site sequences. The mpl promoters controlled by MYPE2900 are one of the smallest genetic switches in bacteria (Fig. 4a). MYPE2900 recombinase and its short recombination site may be utilized as a tool for genetic engineering such as modulation of recombinant gene expressions, inversion or excision of specific chromosomal DNA regions, or construction of gene clones by DNA recombination. To develop these tools, the mpl inversion systems reconstructed in E. coli (with His6-tagged MYPE2900) are useful for detailed characterization of the recombination mechanism and designing of genetic tools.
In our previous study, we did not perform direct detection of mpl promoter inversions in M. penetrans cells (Horino et al., 2003). In the present study, we used a PCR-based method and found the presence of an inverted form of 10 mpl gene promoters (including the p35 and p42 promoters) in M. penetrans genomic DNA. Inversions of these mpl gene promoters occurred at the 12 bp IR sequences that are similar to those of the p35 and p42 promoters (Fig. 4b), suggesting that all these promoter inversions may be catalysed by MYPE2900 recombinase in M. penetrans cells. The 12 bp IR sequences of all mpl genes slightly differ from each other and show the consensus sequence TAAYNNNDATTA. This type of dyad motif is commonly present as a recognition site of site-specific DNA recombinases. However, the 12 bp sequence is considerably shorter than that of the other known recombination sites recognized by site-specific DNA recombinases (Glew et al., 2002; Honarvar et al., 2003; Li et al., 2002; Lysnyansky et al., 2001; Sitaraman et al., 2002). The variation in 12 bp IRs of the mpl gene promoters may contribute to the prevention of inappropriate recombination between different mpl gene promoters and to maintaining the structure of the mpl gene cluster (Fig. 5a). The variation in recombination sites may also affect the frequency of inversion of individual promoters. In this study, we did not examine the inversion frequency of each promoter. This could be evaluated by a real-time PCR method or by using reconstruction systems in M. pneumoniae and E. coli cells in further studies.
Another characteristic feature of the mpl promoter inversion system is the location of the recombinase gene. In many site-specific recombination systems, which control antigenic variations, the Tsr family genes tend to be located near the recombination sites (Chopra-Dewasthaly et al., 2008; Komano, 1999; Kuwahara et al., 2004; Ron et al., 2002). However, in the mpl promoter inversion system, the MYPE2900 gene is located approximately 500 kb apart from the major mpl gene cluster. The other two minor mpl gene clusters are also separated by approximately 600 or 40 kb from the MYPE2900 gene (Horino et al., 2003). In addition, MYPE2900 recombinase efficiently catalysed the inversions of the p42 and p35 promoters in a trans-acting manner in E. coli cells (the MYPE2900 gene and the p42 or p35 promoters were present in different plasmids). Probably, there is no cis-acting effect between the MYPE2900 gene and mpl promoter recombination sites.
The amino acid sequence of MYPE8180 strongly suggests that it is also a Tsr. However, in our experimental conditions, the MYPE8180 product did not show recombinase activity in p42 and p35 promoter inversions. The reconstruction systems in this study are not identical to the natural context in M. penetrans cells, so that there are still possibilities that MYPE8180 recombinase takes part in the mpl system with additional factors in M. penetrans cells or that the His6-tag fused to MYPE8180 in this study disturbed the recombinase activity. However, it is also possible that MYPE8180 has no activity in mpl promoter inversions, but is involved in another site-specific recombination system of the M. penetrans genome. It is known that several mycoplasma species such as Mycoplasma genitalium and M. pneumoniae lack xer-like site-specific recombinase genes, possibly because of reductive evolution of their minimum genomes (approx. 580 and 816 kb in size) (Himmelreich et al., 1997). This fact suggests that these mycoplasma species, as a result of reductive evolution, do not require Xer-like recombinase for resolving their dimer chromosomes during cell division (Glew et al., 2002; Recchia & Sherratt, 1999). However, the genome of M. penetrans (1.36 Mb) is approximately twice as large as that of these species. Furthermore, MYPE8180 shows strong homology to the XerC/D group of the Tsr family proteins. Thus, there is a possibility that MYPE8180 is involved in a Xer-like site-specific recombination system for chromosome segregation in M. penetrans. There is a further possibility that MYPE8180 is involved in another phase or antigenic variation system. To examine these possibilities, identification of the target sequence of MYPE8180 would be an interesting future study.
Identification of MYPE2900 recombinase in this study may lead to further research on disruption of the MYPE2900 gene and development of phase-variation-deficient (phase-locked) strains of M. penetrans. Such phase-locked strains were recently developed in M. agalactiae, a ruminant pathogen that has a multiple site-specific recombination system responsible for its surface lipoprotein variation (Chopra-Dewasthaly et al., 2008). Phase-locked strains are useful for investigating host–pathogen interactions and for determining the functions of each lipoprotein variant. Although a gene disruption method needs to be developed in M. penetrans, an MYPE2900-deficient mutant might be developed in future studies. These attempts may provide an insight into the extent to which surface antigenic variations of mycoplasmas are involved in host immune evasion and help to establish their parasitic lifestyle. An understanding of these pathogenic mechanisms of mycoplasmas may be helpful for designing strategies to eradicate chronic mycoplasma infection.
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ACKNOWLEDGEMENTS |
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Edited by: C. Citti
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Received 27 October 2008;
revised 6 January 2009;
accepted 8 January 2009.
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